Plant-Microbe InteractionsPositive and negative interactions take place not only between microbes but also between microbes and plants. The rhizosphere is a zone of predominantly commensal and mutualistic interactions between plants and microbes. Ecto- and endomycorrhizal fungi provide plants with mineral nutrients and water, receiving photosynthates in return. Under harsh conditions, this mutualistic association can be essential for plant survival. The associations of Dinitrogen-fixing bacteria with certain plants provide essential combined nitrogen for crops and ecosystems. The aerial surfaces of plants provide habitats for largely commensal microbes. On the negative side, certain viruses, bacteria, and fungi cause plant diseases that can result in great economic losses and even severe food shortages.

Some of the positive interactions among plants and microbes are:

Synergistic interactions which include rhizosphere, rhizoplane, phyllosphere and spermosphere.

Mutualistic interactions which include root nodule interactions, leaf nodule interactions and mycorrhizal interactions.

Parasitism is the only negative interaction among the plant and the microbes.

SpermosphereIt is the volume of soil that surrounds a seed. It is an area of increased microbial activity around a germinating seed because of the nutrients leaked into the soil by the germinating seeds. In 1904, M. Duggali was the first to report about the bacterial flora carried by healthy seeds. While most of the microbes are harmless, some may be positively beneficial and some of them may be pathogenic.

Spermosphere organisms forming the normal flora around a germinating seed have some beneficial effects through biological products like growth hormones. Germinating seeds excrete certain chemicals which may influence the quality and quantity of microbes in the vicinity of the seed.

Spermosphere EffectWhen a seed is sown in soil, certain interactions take place between the seed-borne microflora (due to the secretion of certain chemicals by the seed) and the soil-borne microflora which influence the quality of the spermosphere at that condition. When the seed is pre-treated with a fungicide or with any other biological agent, this influences such interactions to a great extent, as for example, the fungicide may totally alter the seed microflora (by inhibiting some fungal flora and increasing some other bacterial flora). This could also influence the nature of microflora that is about to colonise the root (rhizosphere) once the radical emerges out of the seed. Thus by manipulating the spermosphere, one changes the rhizosphere also.

When a seed carrying a natural or altered (by manipulating) load of microbes is sown, certain microbes are activated and others are suppressed. Usually, the microbes that are artificially loaded onto the seed are more dominant flora of the seed and this enables the scientists to beneficially alter the spermosphere of a particular seed, as in Rhizobium, Azotobacter and Azospirillum coated seeds. These organisms get established on the root surface of the germinating seed and benefit the plant (by fixing nitrogen directly into the roots).

Along with spermosphere microflora, the soil-borne flora may also get activated and compete with the former (for nutrition and space). The qualities of chemicals excreted by the germinating seed decides the final quality and quantity of the microflora around the seed. Usually microbes move from the spermosphere to the rhizosphere within three days. Various chemical treatments of the seed (organo-mercurial pesticides) definitely change the rhizosphere micro flora of the seedling thus indicating the plant-root-microbes interactions in the soil through the seed.

When the seed is internally or externally infected by certain pathogenic microorganisms (smut spores), this definitely alters the quality and quantity of the spermosphere and rhizosphere microflora (again through competition). When such a suspected seed is pre-treated with plant protection chemicals, the competition is eliminated (since the pathogen gets killed) and hence the seed gets coated with harmless microbes.

When the seed is pre-treated with organic manure (cow dung) where the seed gets coated with saprophytes present in the manure there is competition between pathogens and non-pathogens (saprophytes present in the organic manure) and depending on the efficiency of one group, one is suppressed and gets eliminated. For example, a seed-borne pathogen of cotton Xanthomonas campestris pv malvacearum is controlled when the seed is pretreated with cow dung slurry containing a lot of saprophytes.

To understand the effect better, we can take this example. When a seed infected with a pathogen is sown in unsterile and sterile soil, there is intense spermospheric effect (enough to suppress the pathogen) in the former whereas in the latter case, pathogen becomes highly virulent (since there is no competition by other organisms).

RhizosphereRhizosphere is the region where soil and roots of the plants make contact or the thin layer of soil adhering to a root system after shaking and removing the loose soil.

Rhizosheath is a modification of rhizosphere, characterised. by a relatively thick soil cylinder that adheres to the plant roots. This is typical of some desert grasses.

Rhizoplane or root surface When the roots are cleaned of all the soil particles adhering to it and then plated, microorganisms can be seen developing indicating that there are certain microbes intimately associated with the root surface. Some fungi inhabit the root surface in a mycelial state, e.g. Cephalosporium, Trichoderma, Penicillium. Specific bacteria also get embedded on the surface of the root with the help of mucilaginous external layer normally present in the actively growing root system.

Rhizosphere effect The direct influence of plant roots on microbes and microbes on plant roots within the rhizosphere is known as the rhizosphere effect.

Effect of Plant Root on Microbial PopulationsThe structure of the plant root system contributes to the establishment of the rhizosphere microbial population. The interactions of plant roots and rhizosphere microorganisms are based largely on interactive modification of the soil environment by processes such as water uptake by the plant and

release of organic chemicals by the roots. The influence of the plant root on the microflora is governed by root exudates, physical and chemical factors in the soil.

Effect of root exudatesThis is the major factor that governs the microflora of the rhizosphere. The root exudates include:

Simple sugars such as glucose and fructose

Di, tri and oligo saccharides

All common amino acids - alanine, serine, leucine, valine, glutamic and asparitic acids. Of these, glutamine and asparagine are produced in large amounts.

Vitamin¾thiamine and biotin

Nucleotides

Flavones and auxins

Stimulators / inhibitors of particular microbes

All these root exudates have an effect on the rhizosphere microflora. Some of the root exudates like the amino acids, promote the growth of microflora of the rhizosphere.

Some nitrogen fixers such as Azospirillum, Azotobacter paspali use the root exudates as the energy source for significant nitrogen fixation.

Thus there is a distinct selective influence of the root system over the microbes. For example, there is a preferential stimulation of gram negative non-spore forming rods in the root region.

Root exudates contaihing toxic substances such as glycosides and hydrocyanic acid may inhibit the growth of pathogens.

One of the attributes of root exudates is the possible role they play in neutralising the soil pH and altering the microclimate of the rhizosphere through liberation of water and CO2, Such changes may influence infections of roots by pathogenic fungi.

Effect of plant growth on rhizosphere microflora The rhizosphere microflora may undergo successional changes as the plant grows from seed germination to maturity. During plant development, a distinct rhizosphere succession results in rapidly growing, growth factor-requiring, opportunistic microbial population. These successional changes correspond to changes in the materials released by the plant root to the rhizosphere during plant maturation.

Initially, carbohydrate and mucilaginous exudates from plant roots stimulate the growth of microorganisms rapidly within the grooves on the root surface and within the mucilaginous sheath (rhizoplane). After the plant matures, autolysis of some of the root materials take place and simple sugars

and amino acids are released into the soil. This further stimulates the growth of bacteria with high intrinsic growth rates, e.g. Pseudomonas. As a result of these effects, the rhizosphere microflora consists of higher proportion of gram negative rods and a lower proportion of gram positive rods, cocci and pleomorphic forms. A relatively higher proportion of motile, rapidly growing bacteria are also seen.

Alteration of rhizosphere microflora This may be done by: Soil amendments This refers to the artificial addition of fertilisers with nitrogen, phosphorous and potassium. This depends on the rhizosphere: soil (R:S) ratio and also on the nutritional content of the chemicals in the soil.

Foliar application of nutrients Translocation of photosynthates from leaves to roots is a well known phenomenon and this does not affect the microflora. So when foliar application of antibiotics, growth regulators, pesticides and inorganic nutrients is carried out, a small amount is being released as root exudates and this can either promote the growth of the present microflora or change the microflora to some extent.

Artificial inoculation This is done on seed or soil with preparation containing live microorganisms especially bacteria (bacterisation). This is beneficial, in that this provides an easier way for the establishment of the microbes to the rhizosphere. This is because as the seed is coated with the live microorganisms, as soon as the root evolves, the colonising of the root takes place and establishment of the other microbes is also made possible. Microbial seed inoculants generally used are Azotobacter, Beijerinckia, Rhizobium or phosphorous solubilising microorganisms.

Effect of Rhizosphere Microbial Population on PlantsThe microorganisms have a marked influence on the growth of plants. The plant growth may be impaired due to the absence of appropriate rhizosphere microflora. The microbial population affect the plant growth in various ways:

Promotion of growth This is brought about by the release of growth factors like auxins and gibberellins that promote plant growth. The organisms which release these growth factor include Arthrobacter, Pseudomonas and Agrobacterium. The production of indole acetic acid (IAA), a plant growth hormone by certain group of microorganisms increases the rate of seed germination and development of root hairs. This is seen in wheat seedlings.

Neutralisation of toxic substances This is seen in the case of plants that grow in flooded sediments, e.g. rice plants and other partially submerged plants. In this case, there is production of hydrogen sulphide generated by the sulphate reduction pathway. This hydrogen sulphide is toxic to the plant roots, and this is neutralised by the bacteria Beggiatoa. This is a microaerophilic, catalase negative, sulphide oxidising filamentous bacterium. This acquires the oxygen and catalase enzyme from the rice plant and aids in the oxidation of toxic H2S to harmless sulphur or sulphate, thus protecting the rice roots.

Allelopathic effect Some substances being released by the microbes can have an antagonistic effect. This may allow plants to enter in amensalic relationship with other plants. Some substances or extracellular products of certain microorganisms lead to the growth of other kinds of microorganisms that can provide

a better rhizosphere microflora. These extracellular products can also inhibit the growth of pathogens, thus protecting the plant roots from getting damaged.

Nutritional recycling The nutrients in the soil are made available to the plants by mobilisation of the nutrients, by fixing it in soil in proper way. Sometimes, the nutrients are made unavailable by immobilisation. For example, microorganisms produce extracellular amino acids, vitamins, etc. using the nutrients and nitrogen fixation process. Thus they make nitrogen available to plants as nitrates or other inorganic forms, e.g. Rhizobium and Azotobacter. Similarly, sulphur oxidisers make sulphur available as sulphates, e.g. Desulfovibrio. Phosphorous is made available as phosphates by the production of acids by the microflora. Siderophore production is another important characteristic feature of rhizosphere microflora. Siderophore production Many microorganisms respond to a fall in the availability of iron in soil by producing extracellular low molecular weight iron transporting agents known as siderophores. These siderophores selectively complex with iron and supply the element to the living cell. They also act as growth factors or antibiotics. For example, Pseudomonas fluorescence (one strain produced a siderophore compound pseudobactin) inhibits the growth of a pathogen Eewinia carotovora by chelating iron from the vicinity of the pathogen and thus reducing the disease severity.

Thus microorganisms increase the recycling and solubilisation of mineral nutrients and making it available to plants. The abundant growth of microbial population in the rhizosphere can sometimes create a deficiency of required minerals for the plants, e.g. bacterial immobilisation of zinc and oxidation of manganese cause the plant diseases 'little leaf' of fruit trees and' gray speck' of oats. Nitrogen is immobilised in the form of microbial protein and some may be lost to the atmosphere by denitrification.

PhyllosphereThe Dutch microbiologist Ruinen coined the term phyllosphere which is the interrelationship between plant foliage and the quality and quantity of microorganisms found on the surface.

The leaf surface is termed as phylloplane. The quality and quantity of the microorganisms on the leaf surface differs with age of .the plant, leaf area, morphology, atmospheric factors (temperature, humidity, etc.).

Growing seasons may also influence the phyllosphere microflora. It increases and reaches the maximum in autumn when the leaves severe. The position of the leaves also plays a role in determining the microflora.

Leaves at the lower levels harbour more microorganisms since they are sheltered and get more nutrients from the raindrops from upper levels.

Plant leaves are exposed to dust and air currents resulting in the establishment of a typical flora on their surface aided by cuticle, waxes and appendages (thorns, spikes) which help in the anchorage of microbes. The leaf diffusates/ exudates promote/ deter the growth of microbes on their surface. The principal nutritive factor in the leaf are amino acids, glucose, fructose and sucrose.

The dominant microorganisms in a forest vegetation are the nitrogen fixing bacteria such as Beijerinckia and Azotobacter. Other genera like Pseudomonas, Enwinia, Sarcina have been encountered in the phyllosphere.

Under damp conditions, some leaves may harbour cyanobacteria like Anabaena, Calothrix, Nostoc and Tolypothrix on their surfaces. Some of the fungi and actinomycetes encountered are Cladosporium,Alternaria, Cercospora, Helminthosporium, Mucor and Streptomyces species.

Characteristic Features of Phyllosphere MicrofloraLeaf surface microbes may perform an effective function in controlling the spread of airborne microbes inciting plant diseases.

Presence of a fungal spore on the surface of leaves incite the formation of a chemical substance referred to as phytoalexin which are active in host defence mechanisms.

Resistance to disease causing microbes has also been attributed to fungistatic compounds secreted by leaves such as malic acid from leaves of Cicer arietinum.

The name elicitor has been commonly used to denote the compounds which induce the synthesis of phytoalexins. These are biotic elicitors such as polysaccharides from fungal cell walls, lipids, microbial enzymes and polypeptides.

Abiotic elicitors are heavy metal salts, detergents, UV light, etc.

Epiphytic microbes are known to synthesise indole acetic acid.

PhylIosphere bacteria are often pigmented due to direct solar radiation.

Pink-pigmented facultative methylotrophs are common in the phyllosphere.

Bacteria can serve as ice nucleators, promoting frost damage to plants. Genetically modified Pseudomonas syringaea lacks a membrane protein that promotes nucleation. Inoculating crops with this organism can lower the temperature at which frost damage occurs.

Azolla-Anabaena symbiosis is a N2-fixing association where cyanobacteria live on leaf surface.

Any change in phyllosphere affects plant growth which in turn affects the physiological activity of root system. Such changes in the root result in an altered pH and spectrum of chemical exudation causing a change in rhizosphere microflora. Thus there is a link between phyllosphere microflora and rhizosphere microflora. There is a continuous diffusion of plant metabolites from the leaves which support the microbial growth and in turn these microbes protect the plant from pathogens.

There are reports on suppression of phyllosphere microflora due to environmental pollution caused especially by cement and fertiliser industries.

Positive Interactions of Plants And MicrobesBacterial mutualistic interactions with plants have been seen in the leaf nodules. Fungal mutualistic interactions are pronounced in plants.

1. Leaf Nodule

2. Mycorrhiza

3.Vam Fungi

Leaf NoduleSymbiotic association of certain bacterial endophytes with leaves of certain plants (usually belonging to the families Rubiaceae and Myrsinaceae) leads to the formation of nodule like structures on the leaves. The plants Psychotria, Pavetta, Chomelia have received considerable importance as leaf nodule producing plants. Isolates of bacteria that have gained importance in formation of leaf nodules are Mycobacterium rubiacearum, Mycoplana rubra, Flavobacterium species, Bacterium rubiacearum, Phyllobacterium rubiacearum and Klebsiella rubiacearum.

There are not many advantages (for the plants) resulting from this sort of mutualistic interactions apart from the fact that the bacterial partners secrete phytohormones (cytokines) for the growth of the plants. The plants definitely provide shelter and photosynthates for the survival of their bacterial partners. Since they form a stable phyllospheric microflora with the plants, these bacterial partners may also prevent the entry of pathogenic spores from entering through the leaves and establishing themselves.

MycorrhizaThe term' mycorrhiza' (literally, fungus root) was first used by A. B.Frank to characterise the association between higher plants and fungi. The symbiotic association between the roots of some plants and some fungi is called mycorrhizal association. The fungus is highly habitat limited and is usually found in the immediate vicinity of or within the roots.

Occurrence of MycorrhizaThey occur on almost all terrestrial plants though not as specific as the nitrogen fixing symbiosis. Thus a plant may have several mycorrhizae that can form symbiosis with its roots. Extent of symbiosis depends on fertility. High soil fertility leads to low mycorrhizal infection and poor symbiosis and vice versa. Roots supply carbohydrates to the fungi which absorb nutrients form the soil and supply them to the crop.

Types of mycorrhizaeMycorrhizae are of two kinds:

Ectomycorrhiza Ectomycorrhizal symbiosis is a mutually beneficial union between fungi and the roots of vascular and non-vascular plants. The host of an ecomycorrhizal fungi is usually a gymnosperm (pine). The typical ectomycorrhizal fungi are basidiomycetes (Agaricus), ascomycetes or phycomycetes members. Usuallly more common in the temperate regions, they are capable of growing apart from the host on media containing simple sugars and vitamins. Ectomycorrhizae have poor competitive saprophytic ability hence they have a tough time competing with other microbes in the soil.

During the infection process, ectomycorrhizal fungi in soil are stimulated by the root metabolites to grow toward the root. The hyphae aggregate around the root and penetrate between the root epidermis and the cortex. A structure called Hartig net is formed which is a fungal sheath surrounding the root in which fungal hyphae penetrate between the root cells. Eventually, the root gets surrounded by a fungal mantle. The fungal hyphae replace the fine lateral root hairs of the host root system thus modifying it structurally. Thus the host root system that is infected with ectomycorrhiza looks stunted and dichotomously branched. The fungal hyphae on the exterior of the roots usually serve as an extension of roots and store large amounts carbohydrates

Endomycorrhiza In this case, the fungal hyphae penetrate the host root cells. They are quite common among the Ericaceae and Orchidaceae members of higher plants as well as fruit trees like citrus, coffee, rubber, etc.

Vam FungiVesicular-arbuscular mycorrhiza (VAM) fungi shown in are geographically ubiquitous. They are commonly found in association with agricultural crops, shrubs, tropical tree species and some temperate trees. Their nutritional requirements are not specific. VAM associations are formed by non septate Zygomycetes and Phycomycetes .fungi. Some examples are Glomus, Gigaspora, Acaulospora, Entrophospora and Scutellospora of which Glomus is the most common fungus.

The fungi are obligate biotrophs and do not grow on synthetic media and hence are classified according to the morphological characteristics of the spores formed in the soil. VAM fungi produce large resting spores (0.2 mm). The spores can survive adverse conditions. The germ tube dies if it is unable to encounter and successfully penetrate the host root. The fungal hyphae traverses and ramifies in the root cortex. Branches from the intercellular hyphae enter cortical cells where further branching results in highly branched hyphal structures called arbuscles.

They are short-lived and serve as the nutrient transfer

Mechanism between the fungus and the host.

Phosphate transfer possibly occurs across living membranes of the host and the fungus via arbuscles. When the association is well established, hyphal swellings called vesicles form on the mycelium inside and outside the root. The vesicles are sac-like terminal swellings at the tip of hyphae and contain many lipid droplets and function as storage organs. External mycelium form very thick-walled chlamydospores.

These fungi are disseminated both actively and passively. Active dissemination is form one root to another by mycelial growth through soil and passive dissemination is through biotic agents like rodents, worms, insects and birds or abiotic agents like wind and water.

VAM fungi interact with other soil microbes like the free-living and symbiotic nitrogen fixers and phosphate solubilisers to improve their efficiency for the biochemical cycling of elements to the host plants.

(a) Non-mycorrhizal conifer root

(b) Mycorrhizal conifer root

Forked short roots the diagram show forked short roots caused by an ectomycorrhizal fungus

Negative Interaction Between Plants And MicrobesAs already discussed, parasitism is the only negative interaction between the plants and microbes.As parasites, the microbes, like bacteria, fungi, viruses and algae, cause infections in the host plant leading the development of disease and loss of commercial value in case the host plant is an agricultural crop.

To cause the disease, the parasite must accomplish two important things:

1. It must enter the host plant.2. It must establish itself at the specific target site within the plant.

After accomplishing this, the parasite is able to overcome the plant defence mechanisms and causes the disease.

There are different portals of entry for each microbe. Thus bacteria and viruses enter the host tissue through the natural openings like stomata, lenticels, etc. Fungi have a separate mode of entry. Either the fungal spore enters directly through the natural openings and germinates within the host tissue, or the spore on falling on the plant surface, forms a special adaptation called appresorium which anchors the spore onto the substratum.

From the appresorium arises a small projection called the infection peg which has a sharp tip through which it penetrates the host cell wall actively

The pathogens can also enter through open wounds (scratches) on the plant surfaces.

Once the pathogen enters the host tissue, it tries to overcome the host defence mechanisms (production of phytoalexins on induction by the elicitors which are the pathogens themselves) and establishes relationship with the host. This interaction is of two kinds. One is biotrophic interaction.

Here the pathogen enters into a harmonious relationship with the host with the pretext that it continues to obtain nutrition from the plant for a long time. Thus it does not kill the plant instantaneously. Another relationship is the necrotrophic wherein the pathogen on entering the host kills the host and obtains the nutrition from the dead tissues.

After the disease is established and even during the course of the disease establishment, a series of symptoms can be visualised on the host surface which is an indication of disease onset.

Some examples of parasites on plants are:

Bacteria¾Enwinia carotovora causing soft rot in carrots; Xanthomonas campestris pv oryzae causing bacterial blight in rice.

Fungus-Fusarium oxysporum causing wilt disease in many plants (cotton, banana).

Virus-Tobacco mosaic virus causing mosaic disease in tobacco.